A microtiter peg lid with ziggurat geometry for medium-throughput antibiotic testing and imaging of biofilms.

Bacteria biofilm responses to disinfectants and antibiotics are quantified and observed using multiple methods, though microscopy, particularly confocal laser scanning microscopy (CLSM) is preferred due to speed, a reduction in user error, and analysis. CLSM can resolve biological and spatial heterogeneity of biofilms in 3D with limited throughput. The microplate peg-lid-based assay, described in ASTM E2799-22, is a medium-throughput method for testing biofilms but does not permit imaging. Breaking off the peg, as recommended by the manufacturer, risks sample damage, and is limited to easily accessible pegs. Here we report modifications to the peg optimized for visualization and visualization of all pegs. We report similar antibiotic challenge recovery via colony formation following the ASTM E2799-22 protocol and imaging. We report novel quantifiable effects of antibiotics on biofilm morphologies, specifically biofilm streamers. The new design bridges the MBEC® assays design that selects for biofilm phenotypes with imaging needs.